It has been known for some time that the polynucleotide polydT will bind to the polydA-polydT duplex to form a colinear triplex (Arnott, S & Selsing E. (1974) J. Molec. Biol. 88, 509). The structure of that triplex has been deduced from X-ray fiber diffraction analysis and has been determined to be a colinear triplex (Arnott, S & Selsing E. (1974) J. Molec. Biol. 88, 509 ). The polydT strand is bound in the parallel orientation to the polydA strand of the underlying duplex. The polydT-polydA-polydT triplex is stabilized by T-A Hoogstein base pairing between A in the duplex and the third strand of polydT. That interaction necessarily places the third strand, called a ligand, within the major groove of the underlying duplex. The binding site in the major groove is also referred to as the target sequence.
Similarly, it has been shown that polydG will bind by triplex formation to the duplex polydG-polydC, presumably by G-G pairing in the major helix groove of the underlying duplex, (Riley M., Mailing B. & Chamberlin M. (1966) J. Molec. Biol. 20, 359). This pattern of association is likely to be similar to the pattern of G-G-C triplet formation seen in tRNA crystals (Cantor C. & Schimmel P., (1980) Biophysical Chemistry vol I, p. 192-195).
Triplexes of the form polydA-polydA-polydT and polydC-polydG-polydC have also been detected (Broitman S., Im D. D. & Fresco J. R. (1987) Proc. Nat. Acad. Sci USA 84, 5120 and Lee J. S., Johnson D. A. & Morgan A. R. (1979) Nucl. Acids Res. 6, 3073). Further the mixed triplex polydCT-polydGA-polydCT has also been observed. (Parseuth D. et al. (1988) Proc. Nat. Acad Sci. USA 85, 1849 and Moser H. E. & Dervan P. B. (1987) Science 238, 645). These complexes, however, have proven to be weak or to occur only at acid PH.
Parallel deoxyribo oligonucleotide isomers which bind in the parallel orientation have been synthesized (Moser H. E. & Dervan P. E. (1987) Science 238, 645-650 and Rajagopol P. & Feigon J. (1989) Nature 339, 637-640). In examples where the binding site was symmetric and could have formed either the parallel or antiparallel triplex (oligodT binding to an oligodA-oligodT duplex target), the resulting triplex formed in the parallel orientation (Moser H. E. & Dervan P. E. (1987) Science 238, 645-650 and Praseuth D. et al (1988) PNAS 85, 1349-1353), as had been deduced from x-ray diffraction analysis of the polydT-polydA-polydT triplex.
Studies employing oligonucleotides comprising the unnatural alpha anomer of the nucleotide subunit, have shown that an antiparallel triplex can form (Praseuth D. et al. (1988) PNAS 85, 1349-1353). However, since the alpha deoxyribonucleotide units of DNA are inherently reversed with respect to the natural beta subunits, an antiparallel triplex formed by alpha oligonucleotides necessarily follows from the observation of parallel triplex formation by the natural beta oligonucleotides. For example, alpha deoxyribo oligonucleotides form parallel rather than antiparallel Watson-Crick helices with a complementary strand of the beta DNA isomer.
It has been demonstrated that a DNA oligonucleotide could bind by triplex formation to a duplex DNA target in a gene control region; thereby repressing transcription initiation (Cooney M. et. al. (1988) Science 241, 456). This was an important observation since the duplex DNA target was not a simple repeating sequence.
The present invention provides a new method for designing synthetic oligonucleotides which will bind tightly and specifically to any duplex DNA target. When the target serves as a regulatory protein the method can be used to design synthetic oligonucleotides which can be used as a class of drug molecules to selectively manipulate the expression of individual genes.